Polypeptides having lactonohydrolase activity and nucleic acids encoding same

ABSTRACT

The present invention relates to isolated polypeptides having lactonohydrolase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides. The present invention further relates to methods for preventing microbial biofilm development.

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a continuation-in-part of pending U.S.application Ser. No. 09/263,041 filed on Mar. 5, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/189,497 filed onNov. 10, 1998, which applications are fully incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to isolated polypeptides havinglactonohydrolase activity and isolated nucleic acid sequences encodingthe polypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing and using the polypeptides.

[0005] 2. Description of the Related Art

[0006] Lactonohydrolases reversibly catalyze the hydrolysis of lactonecompounds to hydroxy acids, i.e., they mediate the interconversionbetween the lactone and acid forms of hydroxy carboxylic acids.

[0007] Shimizu et al. (1992, European Journal of Biochemistry 209:383-390) have disclosed a lactonohydrolase obtained from Fusariumoxysporum. This enzyme preparation stereospecifically hydrolyzesaldonate lactones such as D-galactono-γ-lactone and D-glucono-δ-lactone.In addition, the Fusarium oxysporum lactonohydrolase catalyzes theasymmetric hydrolysis of D-pantoyl lactone, which can be used as achiral building block for the synthesis of D-pantothenate (Shimazu andKataoka, 1996, Annals of the New York Academy of Sciences 799:650-658;Kataoka et al., 1995, Appl. Microbiol. Biotechnol. 44: 333-338; Kataokaet al., 1996, Enzyme Microb. Technol. 19: 307-310). Furthermore,lactonohydrolase irreversibly hydrolyzes a number of aromatic lactones,such as dihydrocoumarin and homogentisic-acid lactone.

[0008] The cloning and expression of a Fusarium oxysporumlactonohydrolase gene has been disclosed (WO 97/10341).

[0009] It is an object of the present invention to provide improvedpolypeptides having lactonohydrolase activity and nucleic acid encodingthe polypeptides.

SUMMARY OF THE INVENTION

[0010] The present invention relates to isolated polypeptides havinglactonohydrolase activity selected from the group consisting of:

[0011] (a) a polypeptide having an amino acid sequence which has atleast 95% identity with amino acids 18 to 400 of SEQ ID NO. 2;

[0012] (b) a polypeptide encoded by a nucleic acid sequence having atleast 95% homology with nucleotides 90 to 1238 of SEQ ID NO. 1;

[0013] (c) a variant of the polypeptide having an amino acid sequence ofSEQ ID NO. 2 comprising a substitution, deletion, and/or insertion ofone or more amino acids;

[0014] (d) an allelic variant of (a) or (b); and

[0015] (e) a fragment of (a), (b), or (d) that has lactonohydrolaseactivity;

[0016] The present invention also relates to isolated nucleic acidsequences encoding the polypeptides and to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing and using the polypeptides.

[0017] The present invention further relates to methods for preventingmicrobial biofilm development.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIGS. 1A and 1B show the cDNA sequence and the deduced amino acidsequence of a Fusarium venenatum lactonohydrolase (SEQ ID NOS. 1 and 2,respectively).

[0019]FIG. 2 shows a restriction map of pDM181.

[0020]FIG. 3 shows a restriction map of pSheB1.

[0021]FIG. 4 shows a restriction map of pTriggs1.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Polypeptides Having Lactonohydrolase Activity

[0023] The term “lactonohydrolase activity” is defined herein as ahydrolase activity which catalyzes the hydrolysis of aldonate andaromatic lactones to the corresponding carboxylic acids. For purposes ofthe present invention, lactonohydrolase activity is determined accordingto the procedure described by Fishbein and Bessman, 1966, Journal ofBiological Chemistry 241: 4835-4841, where the hydrolysis ofD-galactono-γ-lactone is measured.

[0024] In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 18 to 400 of SEQ ID NO. 2 (i.e., the maturepolypeptide) of at least about 95%, and preferably at least about 97%,which have lactonohydrolase activity (hereinafter “homologouspolypeptides”). In a preferred embodiment, the homologous polypeptideshave an amino acid sequence which differs by five amino acids,preferably by four amino acids, more preferably by three amino acids,even more preferably by two amino acids, and most preferably by oneamino acid from amino acids 18 to 400 of SEQ ID NO. 2. For purposes ofthe present invention, the degree of identity between two amino acidsequences is determined by the Clustal method (Higgins, 1989, CABIOS 5:151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters were Ktuple=1, gap penalty=3, windows=5,and diagonals=5.

[0025] Preferably, the polypeptides of the present invention comprisethe amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof;or a fragment thereof that has lactonohydrolase activity. In a morepreferred embodiment, the polypeptide of the present invention comprisesthe amino acid sequence of SEQ ID NO. 2. In another preferredembodiment, the polypeptide of the present invention comprises aminoacids 18 to 400 of SEQ ID NO. 2, or an allelic variant thereof; or afragment thereof that has lactonohydrolase activity. In anotherpreferred embodiment, the polypeptide of the present invention comprisesamino acids 18 to 400 of SEQ ID NO. 2. In another preferred embodiment,the polypeptide of the present invention consists of the amino acidsequence of SEQ ID NO. 2 or an allelic variant thereof; or a fragmentthereof that has lactonohydrolase activity. In another preferredembodiment, the polypeptide of the present invention consists of theamino acid sequence of SEQ ID NO. 2. In another preferred embodiment,the polypeptide consists of amino acids 18 to 400 of SEQ ID NO. 2 or anallelic variant thereof; or a fragment thereof that has lactonohydrolaseactivity. In another preferred embodiment, the polypeptide consists ofamino acids 18 to 400 of SEQ ID NO. 2.

[0026] A fragment of SEQ ID NO. 2 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence. Preferably, a fragment contains at least 310 aminoacid residues, more preferably at least 340 amino acid residues, andmost preferably at least 370 amino acid residues.

[0027] An allelic variant denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

[0028] In a second embodiment, the present invention relates to isolatedpolypeptides having lactonohydrolase activity which are encoded bynucleic acid sequences which hybridize under very low stringencyconditions, preferably low stringency conditions, more preferably mediumstringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with a nucleic acid probewhich hybridizes under the same conditions with (i) nucleotides 90 to1238 of SEQ ID NO. 1, (ii) the genomic sequence comprising nucleotides90 to 1238 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii), or (iv)a complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F.Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.). The subsequence of SEQ ID NO. 1may be at least 100 nucleotides or preferably at least 200 nucleotides.Moreover, the subsequence may encode a polypeptide fragment which haslactonohydrolase activity. The polypeptides may also be allelic variantsor fragments of the polypeptides that have lactonohydrolase activity.

[0029] The nucleic acid sequence of SEQ ID NO. 1 or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO. 2 or afragment thereof, may be used to design a nucleic acid probe to identifyand clone DNA encoding polypeptides having lactonohydrolase activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, preferably at least 25, and more preferably at least 35 nucleotidesin length. Longer probes can also be used. Both DNA and RNA probes canbe used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

[0030] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having lactonohydrolaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO. 1 or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a labeled nucleic acid probe corresponding to the nucleicacid sequence shown in SEQ ID NO. 1, its complementary strand, or asubsequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

[0031] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO. 2, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO. 1. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO. 1. Inanother preferred embodiment, the nucleic acid probe is the nucleic acidsequence contained in plasmid pFA0576 which is contained in Escherichiacoli NRRL B-30074, wherein the nucleic acid sequence encodes apolypeptide having acid phosphatase activity. In another preferredembodiment, the nucleic acid probe is the mature polypeptide codingregion contained in plasmid pFA0576 which is contained in Escherichiacoli NRRL B-30074.

[0032] For long probes of at least 100 nucleotides in length, very lowto very high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

[0033] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

[0034] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at 5° C.to 10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

[0035] For short probes which are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

[0036] In a third embodiment, the present invention relates to variantsof the polypeptide having an amino acid sequence of SEQ ID NO. 2comprising a substitution, deletion, and/or insertion of one or moreamino acids.

[0037] The amino acid sequences of the variant polypeptides may differfrom the amino acid sequence of SEQ ID NO. 2 or the mature polypeptidethereof by an insertion or deletion of one or more amino acid residuesand/or the substitution of one or more amino acid residues by differentamino acid residues. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

[0038] Examples of conservative substitutions are within the group ofbasic amino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

[0039] In a fourth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ ID NO.2 or the mature polypeptide thereof. The immunochemical properties aredetermined by immunological cross-reaction identity tests by thewell-known Ouchterlony double immunodiffusion procedure. Specifically,an antiserum containing polyclonal antibodies which are immunoreactiveor bind to epitopes of the polypeptide having the amino acid sequence ofSEQ ID NO. 2 or the mature polypeptide thereof are prepared byimmunizing rabbits (or other rodents) according to the proceduredescribed by Harboe and Ingild, In N.H. Axelsen, J. Krøll, and B. Weeks,editors, A Manual of Quantitative Immunoelectrophoresis, BlackwellScientific Publications, 1973, Chapter 23, or Johnstone and Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, 1982(more specifically pages 27-31). A polypeptide having immunochemicalidentity is a polypeptide which reacts with the antiserum in anidentical fashion such as total fusion of precipitates, identicalprecipitate morphology, and/or identical electrophoretic mobility usinga specific immunochemical technique. A further explanation ofimmunochemical identity is described by Axelsen, Bock, and Krøll, InN.H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter10. A polypeptide having partial immunochemical identity is apolypeptide which reacts with the antiserum in a partially identicalfashion such as partial fusion of precipitates, partially identicalprecipitate morphology, and/or partially identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of partial immunochemical identity is described by Bock andAxelsen, In N.H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 11.

[0040] The antibody may also be a monoclonal antibody. Monoclonalantibodies may be prepared and used, e.g., according to the methods ofE. Harlow and D. Lane, editors, 1988, Antibodies, A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0041] The polypeptides of the present invention have at least 20%,preferably at least 40%, more preferably at least 60%, even morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 100% of the lactonohydrolase activity of the maturepolypeptide of SEQ ID NO. 2.

[0042] A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide is secreted extracellularly.

[0043] A polypeptide of the present invention may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

[0044] A polypeptide of the present invention may be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

[0045] In a preferred embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

[0046] In another preferred embodiment, the polypeptide is anAspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae, Humicola insolens, Humicola lanuginosa, Mucormiehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

[0047] In another preferred embodiment, the polypeptide is a Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum polypeptide.

[0048] In a more preferred embodiment, the Fusarium venenatum cell isFusarium venenatum A3/5, which was originally deposited as Fusariumgraminearum ATCC 20334 and recently reclassified as Fusarium venenatumby Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; aswell as taxonomic equivalents of Fusarium venenatum regardless of thespecies name by which they are currently known. In another preferredembodiment, the Fusarium venenatum cell is a morphological mutant ofFusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosedin WO 97/26330.

[0049] It will be understood that for the aforementioned species, theinvention encompasses both the perfect and imperfect states, and othertaxonomic equivalents, e.g., anamorphs, regardless of the species nameby which they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents. For example, taxonomicequivalents of Fusarium are defined by D. L. Hawksworth, P. M. Kirk, B.C. Sutton, and D. N. Pegler (editors), 1995, In Ainsworth & Bisby'sDictionary of the Fungi, Eighth Edition, CAB International, UniversityPress, Cambridge, England, pp.173-174.

[0050] Strains of these species are readily accessible to the public ina number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

[0051] Furthermore, such polypeptides may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

[0052] As defined herein, an “isolated” polypeptide is a polypeptidewhich is essentially free of other non-lactonohydrolase polypeptides,e.g., at least about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

[0053] Polypeptides encoded by nucleic acid sequences of the presentinvention also include fused polypeptides or cleavable fusionpolypeptides in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide or fragment thereof. A fusedpolypeptide is produced by fusing a nucleic acid sequence (or a portionthereof) encoding another polypeptide to a nucleic acid sequence (or aportion thereof) of the present invention. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator.

[0054] Nucleic Acid Sequences

[0055] The present invention also relates to isolated nucleic acidsequences which encode a polypeptide of the present invention. In apreferred embodiment, the nucleic acid sequence is set forth in SEQ IDNO. 1. In another more preferred embodiment, the nucleic acid sequenceis the sequence contained in plasmid pFA0576 that is contained inEscherichia coli NRRL B-30074. In another preferred embodiment, thenucleic acid sequence is the mature polypeptide coding region of SEQ IDNO. 1. In another more preferred embodiment, the nucleic acid sequenceis the mature polypeptide coding region contained in plasmid pFA0576that is contained in Escherichia coli NRRL B-30074. The presentinvention also encompasses nucleic acid sequences which encode apolypeptide having the amino acid sequence of SEQ ID NO. 2 or the maturepolypeptide thereof, which differ from SEQ ID NO. 1 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO. 1 which encode fragments of SEQ ID NO. 2 thathave lactonohydrolase activity.

[0056] A subsequence of SEQ ID NO. 1 is a nucleic acid sequenceencompassed by SEQ ID NO. 1 except that one or more nucleotides from the5′ and/or 3′ end have been deleted. Preferably, a subsequence containsat least 930 nucleotides, more preferably at least 1020 nucleotides, andmost preferably at least 1110 nucleotides.

[0057] The present invention also relates to mutant nucleic acidsequences comprising at least one mutation in the mature polypeptidecoding sequence of SEQ ID NO. 1, in which the mutant nucleic acidsequence encodes a polypeptide which consists of amino acids 18 to 400of SEQ ID NO. 2.

[0058] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, New York. Other nucleic acid amplification procedures such asligase chain reaction (LCR), ligated activated transcription (LAT) andnucleic acid sequence-based amplification (NASBA) may be used. Thenucleic acid sequence may be cloned from a strain of Fusarium, oranother or related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of the nucleic acidsequence.

[0059] The term “isolated nucleic acid sequence” as used herein refersto a nucleic acid sequence which is essentially free of other nucleicacid sequences, e.g., at least about 20% pure, preferably at least about40% pure, more preferably at least about 60% pure, even more preferablyat least about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

[0060] The present invention also relates to nucleic acid sequenceswhich have a degree of homology to the mature polypeptide codingsequence of SEQ ID NO. 1 (i.e., nucleotides 90 to 1238) of at leastabout 95%, and preferably about 97% homology, which encode an activepolypeptide. For purposes of the present invention, the degree ofhomology between two nucleic acid sequences is determined by theWilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of theNational Academy of Science USA 80: 726-730) using the LASERGENE™MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10 andgap length penalty of 10. Pairwise alignment parameters were Ktuple=3,gap penalty=3, and windows=20.

[0061] Modification of a nucleic acid sequence encoding a polypeptide ofthe present invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO. 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

[0062] It will be apparent to those skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidresidues essential to the activity of the polypeptide encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for lactonohydrolase activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labelling (see, e.g., de Vos et a., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904;Wlodaver et a., 1992, FEBS Letters 309: 59-64).

[0063] The present invention also relates to isolated nucleic acidsequences encoding a polypeptide of the present invention, whichhybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with the nucleic acid sequence of SEQ ID NO. 1 or itscomplementary strand; or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

[0064] The present invention also relates to isolated nucleic acidsequences produced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 90 to 1238 of SEQ ID NO. 1, (ii) the genomic sequencecomprising nucleotides 90 to 1238 of SEQ ID NO. 1, (iii) a subsequenceof (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii);and (b) isolating the nucleic acid sequence. The subsequence ispreferably a sequence of at least 100 nucleotides such as a sequencewhich encodes a polypeptide fragment which has lactonohydrolaseactivity.

[0065] Methods for Producing Mutant Nucleic Acid Sequences

[0066] The present invention further relates to methods for producing amutant nucleic acid sequence, comprising introducing at least onemutation into the mature polypeptide coding sequence of SEQ ID NO. 1 ora subsequence thereof, wherein the mutant nucleic acid sequence encodesa polypeptide which consists of amino acids 18 to 400 of SEQ ID NO. 2 ora fragment thereof which has lactonohydrolase activity.

[0067] The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

[0068] Nucleic Acid Constructs

[0069] The present invention also relates to nucleic acid constructscomprising a nucleic acid sequence of the present invention operablylinked to one or more control sequences which direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Expression will be understood to include anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

[0070] “Nucleic acid construct” is defined herein as a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid combined and juxtaposed in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a nucleic acid sequence which directly specifies the aminoacid sequence of its protein product. The boundaries of the codingsequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

[0071] An isolated nucleic acid sequence encoding a polypeptide of thepresent invention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

[0072] The term “control sequences” is defined herein to include allcomponents which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide. The term “operably linked” is definedherein as a configuration in which a control sequence is appropriatelyplaced at a position relative to the coding sequence of the DNA sequencesuch that the control sequence directs the expression of a polypeptide.

[0073] The control sequence may be an appropriate promoter sequence, anucleic acid sequence which is recognized by a host cell for expressionof the nucleic acid sequence. The promoter sequence containstranscriptional control sequences which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

[0074] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1 983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0075] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention in a filamentousfungal host cell are promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787),as well as the NA2-tpi promoter (a hybrid of the promoters from thegenes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

[0076] In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

[0077] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

[0078] Preferred terminators for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

[0079] Preferred terminators for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), andSaccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

[0080] The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

[0081] Preferred leaders for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

[0082] Suitable leaders for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase,Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0083] The control sequence may also be a polyadenylation sequence, asequence operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

[0084] Preferred polyadenylation sequences for filamentous fungal hostcells are obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

[0085] Useful polyadenylation sequences for yeast host cells aredescribed by Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

[0086] The control sequence may also be a signal peptide coding regionthat codes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

[0087] Effective signal peptide coding regions for bacterial host cellsare the signal peptide coding regions obtained from the genes forBacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0088] Effective signal peptide coding regions for filamentous fungalhost cells are the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0089] In a preferred embodiment, the signal peptide coding region isnucleotides 38 to 89 of SEQ ID NO. 1 which encodes amino acids 1 to 17of SEQ ID NO. 2.

[0090] Useful signal peptides for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions aredescribed by Romanos et al., 1992, supra.

[0091] The control sequence may also be a propeptide coding region thatcodes for an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

[0092] Where both signal peptide and propeptide regions are present atthe amino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

[0093] It may also be desirable to add regulatory sequences which allowthe regulation of the expression of the polypeptide relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

[0094] The present invention also relates to nucleic acid constructs foraltering the expression of an endogenous gene encoding a polypeptide ofthe present invention. The constructs may contain the minimal number ofcomponents necessary for altering expression of the endogenous gene. Inone embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, and (d) asplice-donor site. Upon introduction of the nucleic acid construct intoa cell, the construct inserts by homologous recombination into thecellular genome at the endogenous gene site. The targeting sequencedirects the integration of elements (a)-(d) into the endogenous genesuch that elements (b)-(d) are operably linked to the endogenous gene.In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

[0095] In both embodiments, the introduction of these components resultsin production of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

[0096] The targeting sequence can be within the endogenous gene,immediately adjacent to the gene, within an upstream gene, or upstreamof and at a distance from the endogenous gene. One or more targetingsequences can be used. For example, a circular plasmid or DNA fragmentpreferably employs a single targeting sequence, while a linear plasmidor DNA fragment preferably employs two targeting sequences.

[0097] The regulatory sequence of the construct can be comprised of oneor more promoters, enhancers, scaffold-attachment regions or matrixattachment sites, negative regulatory elements, transcription bindingsites, or combinations of these sequences.

[0098] The constructs further contain one or more exons of theendogenous gene. An exon is defined as a DNA sequence which is copiedinto RNA and is present in a mature mRNA molecule such that the exonsequence is in-frame with the coding region of the endogenous gene. Theexons can, optionally, contain DNA which encodes one or more amino acidsand/or partially encodes an amino acid. Alternatively, the exon containsDNA which corresponds to a 5′ non-encoding region. Where the exogenousexon or exons encode one or more amino acids and/or a portion of anamino acid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

[0099] The splice-donor site of the constructs directs the splicing ofone exon to another exon. Typically, the first exon lies 5′ of thesecond exon, and the splice-donor site overlapping and flanking thefirst exon on its 3′ side recognizes a splice-acceptor site flanking thesecond exon on the 5′ side of the second exon. A splice-acceptor site,like a splice-donor site, is a sequence which directs the splicing ofone exon to another exon. Acting in conjunction with a splice-donorsite, the splicing apparatus uses a splice-acceptor site to effect theremoval of an intron.

[0100] Expression Vectors

[0101] The present invention also relates to recombinant expressionvectors comprising a nucleic acid sequence of the present invention, apromoter, and transcriptional and translational stop signals. Thevarious nucleic acid and control sequences described above may be joinedtogether to produce a recombinant expression vector which may includeone or more convenient restriction sites to allow for insertion orsubstitution of the nucleic acid sequence encoding the polypeptide atsuch sites. Alternatively, the nucleic acid sequence of the presentinvention may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

[0102] The recombinant expression vector may be any vector (e.g., aplasmid or virus) which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

[0103] The vector may be an autonomously replicating vector, i.e., avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

[0104] The vectors of the present invention preferably contain one ormore selectable markers which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

[0105] The vectors of the present invention preferably contain anelement(s) that permits stable integration of the vector into the hostcell's genome or autonomous replication of the vector in the cellindependent of the genome.

[0106] For integration into the host cell genome, the vector may rely onthe nucleic acid sequence encoding the polypeptide or any other elementof the vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

[0107] For autonomous replication, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously inthe host cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pBR322, pUC19, pACYC177, andpACYC184 permitting replication in E. coli, and pUB110, pE 194, pTA1060, and pAMβ1 permitting replication in Bacillus. Examples of originsof replication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes its functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1433).

[0108] More than one copy of a nucleic acid sequence of the presentinvention may be inserted into the host cell to increase production ofthe gene product. An increase in the copy number of the nucleic acidsequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

[0109] The procedures used to ligate the elements described above toconstruct the recombinant expression vectors of the present inventionare well known to one skilled in the art (see, e.g., Sambrook et al.,1989, supra).

[0110] Host Cells

[0111] The present invention also relates to recombinant host cells,comprising a nucleic acid sequence of the invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

[0112] The host cell may be a unicellular microorganism, e.g., aprokaryote, or a non-unicellular microorganism, e.g., a eukaryote.

[0113] Useful unicellular cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausil, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

[0114] The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0115] The host cell may be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

[0116] In a preferred embodiment, the host cell is a fungal cell.“Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

[0117] In a more preferred embodiment, the fungal host cell is a yeastcell. “Yeast” as used herein includes ascosporogenous yeast(Endomycetales), basidiosporogenous yeast, and yeast belonging to theFungi Imperfecti (Blastomycetes). Since the classification of yeast maychange in the future, for the purposes of this invention, yeast shall bedefined as described in Biology and Activities of Yeast (Skinner, F. A.,Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol.Symposium Series No. 9, 1980).

[0118] In an even more preferred embodiment, the yeast host cell is aCandida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

[0119] In a most preferred embodiment, the yeast host cell is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

[0120] In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such asSaccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

[0121] In an even more preferred embodiment, the filamentous fungal hostcell is a cell of a species of, but not limited to, Acremonium,Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,Penicillium, Thielavia, Tolypocladium, or Trichoderma.

[0122] In a most preferred embodiment, the filamentous fungal host cellis an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningli, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0123] Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al, 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al, 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

[0124] Methods of Production

[0125] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide. Preferably, the strain is of the genusFusarium, and more preferably Fusarium venenatum.

[0126] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

[0127] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the mature polypeptide coding region of SEQ ID NO.1, wherein the mutant nucleic acid sequence encodes a polypeptide whichconsists of amino acids 18 to 400 of SEQ ID NO. 2, and (b) recoveringthe polypeptide.

[0128] The present invention further relates to methods for producing apolypeptide of the present invention comprising (a) cultivating ahomologously recombinant cell, having incorporated therein a newtranscription unit comprising a regulatory sequence, an exon, and/or asplice donor site operably linked to a second exon of an endogenousnucleic acid sequence encoding the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The methods are based on the use of gene activationtechnology, for example, as described in U.S. Pat. No. 5,641,670.

[0129] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0130] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide as describedherein.

[0131] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

[0132] The polypeptides of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J. -C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

[0133] Plants

[0134] The present invention also relates to a transgenic plant, plantpart, or plant cell which has been transformed with a nucleic acidsequence encoding a polypeptide having lactonohydrolase activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

[0135] The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). Examples of monocot plants are grasses,such as meadow grass (blue grass, Poa), forage grass such as festuca,lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat,oats, rye, barley, rice, sorghum, and maize (corn).

[0136] Examples of dicot plants are tobacco, legumes, such as lupins,potato, sugar beet, pea, bean and soybean, and cruciferous plants(family Brassicaceae), such as cauliflower, rape seed, and the closelyrelated model organism Arabidopsis thaliana.

[0137] Examples of plant parts are stem, callus, leaves, root, fruits,seeds, and tubers. Also specific plant tissues, such as chloroplast,apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm areconsidered to be a plant part. Furthermore, any plant cell, whatever thetissue origin, is considered to be a plant part.

[0138] Also included within the scope of the present invention are theprogeny of such plants, plant parts and plant cells.

[0139] The transgenic plant or plant cell expressing a polypeptide ofthe present invention may be constructed in accordance with methodsknown in the art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

[0140] Conveniently, the expression construct is a nucleic acidconstruct which comprises a nucleic acid sequence encoding a polypeptideof the present invention operably linked with appropriate regulatorysequences required for expression of the nucleic acid sequence in theplant or plant part of choice. Furthermore, the expression construct maycomprise a selectable marker useful for identifying host cells intowhich the expression construct has been integrated and DNA sequencesnecessary for introduction of the construct into the plant in question(the latter depends on the DNA introduction method to be used).

[0141] The choice of regulatory sequences, such as promoter andterminator sequences and optionally signal or transit sequences isdetermined, for example, on the basis of when, where, and how thepolypeptide is desired to be expressed. For instance, the expression ofthe gene encoding a polypeptide of the present invention may beconstitutive or inducible, or may be developmental, stage or tissuespecific, and the gene product may be targeted to a specific tissue orplant part such as seeds or leaves. Regulatory sequences are, forexample, described by Tague et al., 1988, Plant Physiology 86: 506.

[0142] For constitutive expression, the 35S-CaMV promoter may be used(Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters maybe, for example, a promoter from storage sink tissues such as seeds,potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

[0143] A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

[0144] The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

[0145] The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

[0146] Presently, Agrobacterium tumefaciens-mediated gene transfer isthe method of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

[0147] Following transformation, the transformants having incorporatedtherein the expression construct are selected and regenerated into wholeplants according to methods well-known in the art.

[0148] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having lactonohydrolase activity of the presentinvention under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

[0149] Removal or Reduction of Lactonohydrolase Activity

[0150] The present invention also relates to methods for producing amutant cell of a parent cell, which comprises disrupting or deleting anucleic acid sequence encoding the polypeptide or a control sequencethereof, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

[0151] The construction of strains which have reduced lactonohydrolaseactivity may be conveniently accomplished by modification orinactivation of a nucleic acid sequence necessary for expression of thepolypeptide having lactonohydrolase activity in the cell. The nucleicacid sequence to be modified or inactivated may be, for example, anucleic acid sequence encoding the polypeptide or a part thereofessential for exhibiting lactonohydrolase activity, or the nucleic acidsequence may have a regulatory function required for the expression ofthe polypeptide from the coding sequence of the nucleic acid sequence.An example of such a regulatory or control sequence may be a promotersequence or a functional part thereof, i.e., a part which is sufficientfor affecting expression of the polypeptide. Other control sequences forpossible modification are described above.

[0152] Modification or inactivation of the nucleic acid sequence may beperformed by subjecting the cell to mutagenesis and selecting orscreening for cells in which the lactonohydrolase producing capabilityhas been reduced. The mutagenesis, which may be specific or random, maybe performed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

[0153] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues.

[0154] When such agents are used, the mutagenesis is typically performedby incubating the cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor cells exhibiting reduced lactonohydrolase activity or production.

[0155] Modification or inactivation of production of a polypeptide ofthe present invention may be accomplished by introduction, substitution,or removal of one or more nucleotides in the nucleic acid sequenceencoding the polypeptide or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change of the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleicacid sequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

[0156] An example of a convenient way to eliminate or reduce productionby a host cell of choice is by gene replacement or gene interruption. Inthe gene interruption method, a nucleic acid sequence corresponding tothe endogenous gene or gene fragment of interest is mutagenized in vitroto produce a defective nucleic acid sequence which is then transformedinto the host cell to produce a defective gene. By homologousrecombination, the defective nucleic acid sequence replaces theendogenous gene or gene fragment. It may be desirable that the defectivegene or gene fragment also encodes a marker which may be used forselection of transformants in which the gene encoding the polypeptidehas been modified or destroyed.

[0157] Alternatively, modification or inactivation of the nucleic acidsequence may be performed by established anti-sense techniques using anucleotide sequence complementary to the polypeptide encoding sequence.More specifically, production of the polypeptide by a cell may bereduced or eliminated by introducing a nucleotide sequence complementaryto the nucleic acid sequence encoding the polypeptide which may betranscribed in the cell and is capable of hybridizing to the polypeptidemRNA produced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the polypeptide mRNA, theamount of polypeptide translated is thus reduced or eliminated.

[0158] It is preferred that the cell to be modified in accordance withthe methods of the present invention is of microbial origin, forexample, a fungal strain which is suitable for the production of desiredprotein products, either homologous or heterologous to the cell.

[0159] The present invention further relates to a mutant cell of aparent cell which comprises a disruption or deletion of a nucleic acidsequence encoding the polypeptide or a control sequence thereof, whichresults in the mutant cell producing less of the polypeptide than theparent cell.

[0160] The polypeptide-deficient mutant cells so created areparticularly useful as host cells for the expression of homologousand/or heterologous polypeptides. Therefore, the present inventionfurther relates to methods for producing a homologous or heterologouspolypeptide comprising (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

[0161] In a further aspect, the present invention relates to a methodfor producing a protein product essentially free of lactonohydrolaseactivity by fermentation of a cell which produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibitinglactonohydrolase activity to the fermentation broth before, during, orafter the fermentation has been completed, recovering the product ofinterest from the fermentation broth, and optionally subjecting therecovered product to further purification.

[0162] In a further aspect, the present invention relates to a methodfor producing a protein product essentially free of lactonohydrolaseactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce thelactonohydrolase activity substantially, and recovering the product fromthe culture broth. Alternatively, the combined pH and temperaturetreatment may be performed on an enzyme preparation recovered from theculture broth. The combined pH and temperature treatment may optionallybe used in combination with a treatment with a lactonohydrolaseinhibitor.

[0163] In accordance with this aspect of the invention, it is possibleto remove at least 60%, preferably at least 75%, more preferably atleast 85%, still more preferably at least 95%, and most preferably atleast 99% of the lactonohydrolase activity. Complete removal oflactonohydrolase activity may be obtained by use of this method.

[0164] The combined pH and temperature treatment is preferably carriedout at a pH in the range of 6.5-7.5 and a temperature in the range of40-70° C. for a sufficient period of time to attain the desired effect,where typically, 30 to 60 minutes is sufficient.

[0165] The methods used for cultivation and purification of the productof interest may be performed by methods known in the art.

[0166] The methods of the present invention for producing an essentiallylactonohydrolase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,galactosidase, beta-galactosidase, glucoamylase, glucose oxidase,glucosidase, haloperoxidase, hemicellulase, invertase, isomerase,laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolyticenzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transferase, transglutaminase, orxylanase. The lactonohydrolase-deficient cells may also be used toexpress heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like.

[0167] It will be understood that the term “eukaryotic polypeptides”includes not only native polypeptides, but also those polypeptides,e.g., enzymes, which have been modified by amino acid substitutions,deletions or additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

[0168] In a further aspect, the present invention relates to a proteinproduct essentially free from lactonohydrolase activity which isproduced by a method of the present invention.

[0169] Biofilms

[0170] The polypeptides of the present invention may be used forpreventing the development of a microbial biofilm (also known as slime).

[0171] Biofilms are biological films that develop and persist at solidsurfaces in aqueous environments from the adsorption of microbial cellsonto the solid surfaces (Palmer and White, 1997, Trends in Microbiology5: 435-440; Costerton et al., 1987, Annual Reviews of Microbiology 41:435-464; Mueller, 1994, TAPPI Proceedings, 1994 Biological SciencesSymposium 195-201). This adsorption can provide a competitive advantagefor the microorganisms since they can reproduce, are accessible to awider variety of nutrients and oxygen conditions, are not washed away,and are less sensitive to antimicrobial agents. The formation of thebiofilm is also accompanied by the production of exo-polymeric materials(polysaccharides, polyuronic acids, alginates, glycoproteins, andproteins) which together with the cells form thick layers ofdifferentiated structures separated by water-filled spaces (McEldowneyand Fletcher, 1986, Journal of General Microbiology 132: 513-523;Sutherland, Surface Carbohydrates of the Prokaiyotic Cell, AcademicPress, New York, 1977, pp. 27-96). The resident microorganisms may beindividual species of microbial cells or mixed communities of microbialcells, which may include aerobic and anaerobic bacteria, algae,protozoa, and fungi.

[0172] Thus, the biofilm is a complex assembly of living microorganismsembedded in an organic structure composed of one or more matrix polymerswhich are secreted by the resident microorganisms.

[0173] Biofilms can develop into macroscopic structures severalmillimeters or centimeters in thickness and cover large surface areas.These formations can play a role in restricting or entirely blockingflow in plumbing systems, decreasing heat transfer in heat exchangers,or causing pathogenic problems in municipal water supplies, foodprocessing, medical devices (e.g., catheters, orthopedic devices,implants) and often decrease the life of materials through corrosiveaction mediated by the embedded microorganisms. This biological foulingis a serious economic problem in industrial water process systems, pulpand paper production processes, cooling water systems, injection wellsfor oil recovery, cooling towers, porous media (sand and soil), marineenvironments, and air conditioning systems, and any closed waterrecirculation system.

[0174] The removal or prevention of biofilm traditionally requires theuse of dispersants, surfactants, detergents, enzyme formulations,anti-microbials, biocides, boil-out procedures, and/or corrosivechemicals, e.g., base. Procedures for using these measures are wellknown in the art. For example, removal of biofilm build-up in a papermachine in the pulp and paper industry traditionally requires a depositcontrol program including proper housekeeping to keep surfaces free ofsplashed stock, anti-microbial treatment of fresh water and additives,the use of biocides to reduce microbiological growth on the machine, andscheduled boil-outs to remove the deposits that do form.

[0175] The formation of a biofilm by Pseudomonas aeruginosa involves theproduction of at least two extracellular signals involved in cell-tocell communication (WO 98/58075). The two cell-to-cell signaling systemsare the lasR-lasI and rhlR-rhil (also called vsmR-vsml) systems (Davieset al., 1998, Science 280: 295-298). The lasl gene directs the synthesisof a diffusible extracellular signal, N-(3-oxododecanoyl)-L-homoserinelactone. The lasR product is a transcriptional regulator that requiressufficient levels of N-(3-oxododecanoyl)-L-homoserine lactone toactivate a number of virulence genes, including lasi, and the rhlR-rhilsystem. The rhil gene directs the synthesis of the extracellular signal,N-buytryl-L-homoserine lactone, which is required for activation ofvirulence genes and expression of the stationary-phase factor, RpoS, bythe rhlR gene product. This type of gene regulation has been termedquorum sensing and response. Davies et al have demonstrated that thelasR-lasl system was involved in the differentiation of biofilmformation. WO 98/58075 provides a method whereby cell-cell communicationin bacteria via the lasR-lasl system is manipulated to control biofilmarchitecture and structural integrity.

[0176] The present invention also relates to methods for preventingbiofilm development on a liquid-solid interface by one or moremicroorganisms, comprising administering an effective amount of acomposition comprising one or more polypeptides having lactonohydrolaseactivity and a carrier to the liquid-solid interface to degrade one ormore lactones produced by the one or more microorganisms, wherein theone or more lactones are involved in the formation of the biofilm.

[0177] The lactone may be any lactone involved in biofilm formation. Ina preferred embodiment, the lactone is a homoserine lactone. In a morepreferred embodiment, the lactone is N-(3-oxododecanoyl)-L-homoserinelactone. In another more preferred embodiment, the lactone isN-butyryl-L-homoserine lactone.

[0178] The liquid-solid interface may be any system prone to biofilmdevelopment, particularly the systems enumerated above.

[0179] The biofilm may be produced by an integrated community of two ormore micoorganisms or by one microorganism. The microorganism may be anymicroorganism involved in biofilm production including an aerobic oranaerobic bacterium (Gram positive and Gram negative), fungus (yeast orfilamentous fungus), algae, and protozoan. In a preferred embodiment,the bacteria is an aerobic bacterium. In another preferred embodiment,the bacterium is an anaerobic bacterium. In a more preferred embodiment,the aerobic bacterium is a Pseudomonas. In another more preferredembodiment, the aerobic bacterium is a Flavobacterium. In another morepreferred embodiment, the anaerobic bacterium is a Desulfovibrio. In amost preferred embodiment, the aerobic bacterium is Pseudomonasaeruginosa. In another most preferred embodiment, the anaerobicbacterium is Desulfovibrio desulfuricans.

[0180] The composition comprising one or more polypeptides havinglactonohydrolase activity may be a liquid, spray, or powder formulation.The composition may be augmented with one or more agents for degrading,removing, or preventing the formation of the biofilm. These agents mayinclude, but are not limited to, dispersants, surfactants, detergents,enzyme formulations, anti-microbials, and biocides.

[0181] In a preferred embodiment, the agent is a surfactant. In a morepreferred embodiment, the surfactant is sodium dodecyl sulfate,quaternary ammonium compounds, alkyl pyridinium iodides, Tween 80,Tween, 85, Triton X-100, Brij 56, biological surfactants, rhamnolipid,surfactin, visconsin, or sulfonates.

[0182] In a preferred embodiment, the agent is one or more enzymes. In amore preferred embodiment, the one or more enzymes is selected from thegroup consisting of a protease, alginate lyase, and carbohydrase

[0183] The present invention also relates to such compositions forpreventing development of a biofilm. Furthermore, the composition may bea disinfectant composition. The disinfectant composition may be usefulas a disinfectant for Gram negative bacteria from, including but notlimited to, Pseudomonadaceae, Azatobacteraceae, Rhizabiaeceae,Methylococcaceae, Halobacteriaceae, Legionellaceae, Neisseriaceae.

[0184] Other Uses

[0185] The present invention is also directed to other methods of usingthe polypeptides having lactonohydrolase activity.

[0186] The polypeptides of the present invention may be used in thehydrolysis of aldonate γ- or δ-lactones (e.g., D-gulono-γ-lactone,L-mannono-γ-lactone, D-galactono-γ-lactone, or D-glucono-δ-lactone) oraromatic γ- or δ-lactones (e.g., dihydrocoumarin and homogentisic acidlactone) to the corresponding acids.

[0187] The polypeptides of the present invention may be also used forthe optical resolution of D,L-pantolactone via D-selective asymmetrichydrolysis to produce D-pantothenate according to the methods describedin WO 97/10341. However, the enzyme may be cell-bound, immobilized on acarrier, or used in a free form using methods well known in the art forenzymatic optical resolution.

[0188] The polypeptides of the present invention may be also used forthe debittering of citrus juice via the hydrolysis of a aldonate oraromatic γ- or δ-lactone, for example, limonin.

[0189] The polypeptides of the present invention may also be used forthe detoxification of apple juice contaminated with patulin.

[0190] Signal Peptide

[0191] The present invention also relates to nucleic acid constructscomprising a gene encoding a protein operably linked to a nucleic acidsequence consisting of nucleotides 38 to 89 of SEQ ID NO. 1 encoding asignal peptide consisting of amino acids 1 to 17 of SEQ ID NO. 2,wherein the gene is foreign to the nucleic acid sequence.

[0192] The present invention also relates to recombinant expressionvectors and recombinant host cells comprising such a nucleic acidconstruct.

[0193] The present invention also relates to methods for producing aprotein comprising (a) cultivating such a recombinant host cell underconditions suitable for production of the protein; and (b) recoveringthe protein.

[0194] The nucleic acid sequence may be operably linked to foreign geneswith other control sequences. Such other control sequences are describedsupra.

[0195] The protein may be native or heterologous to a host cell. Theterm “protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

[0196] Preferably, the protein is a hormone, hormone variant, enzyme,receptor or a portion thereof, antibody or a portion thereof, orreporter. In a more preferred embodiment, the protein is anoxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. Inan even more preferred embodiment, the protein is an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

[0197] The gene may be obtained from any prokaryotic, eukaryotic, orother source.

[0198] The present invention is further described by the followingexamples which should not be construed as limiting the scope of theinvention.

Examples

[0199] Chemicals used as buffers and substrates were commercial productsof at least reagent grade.

[0200] Media and Solutions

[0201] COVE trace metals solution was composed per liter of 0.04 g ofNaB₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄. 7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂0, and 10 g of ZnSO₄.7H₂O.

[0202] 50×COVE salts solution was composed per liter of 26 g of KCl, 26g of MgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals.

[0203] COVE medium was composed per liter of 342.3 g of sucrose, 20 mlof 50×COVE salt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl₂,and 25 g of Noble agar. 50×Vogels medium was composed per liter of 150 gof sodium citrate, 250 g of KH₂PO₄, 10 g of MgSO₄.7H₂O, 10 g ofCaCl₂.2H₂O, 2.5 ml of biotin stock solution, and 5.0 ml of AMG tracemetals solution.

[0204] COVE top agarose was composed per liter of 20 ml of 50×COVEsalts, 0.8 M sucrose, 1.5 M cesium chloride, 1.0 M acetamide, and 10 gof low melt agarose, pH adjusted to 6.0.

[0205] RA sporulation medium was composed per liter of 50 g of succinicacid, 12.1 g of NaNO₃, 1 g of glucose, 20 ml of 50×Vogels, and 0.5 ml ofa 10 mg/ml NaMoO₄ stock solution, pH to 6.0.

[0206] YEPG medium was composed per liter of 10 g of yeast extract, 20 gof peptone, and 20 g of glucose.

[0207] STC was composed of 0.8 M sorbitol, 25 mM Tris pH 8, 25 mM CaCl₂.

[0208] SPTC was composed of 40% PEG 4000, 0.8 M sorbitol, 25 mM Tris pH8, 25 mM CaCl₂.

[0209] M400Da medium was composed per liter of 50 g of maltodextrin, 2 gof MgSO₄.7H₂O, 2 g of KH₂PO₄, 4 g of citric acid, 8 g of yeast extract,2 g of urea, and 1 ml of COVE trace metals solution.

Example 1

[0210] Fermentation and Mycelial Tissue

[0211]Fusarium venenatum CC1-3, a morphological mutant of Fusariumstrain ATCC 20334 (Wiebe et al., 1991, Mycol. Research 95: 1284-1288),was grown in a two-liter lab-scale fermentor using a fed-batchfermentation scheme with NUTRIOSE™ (Roquette Freres, S. A., Beinheim,France) as the carbon source and yeast extract. Ammonium phosphate wasprovided in the feed. The pH was maintained at 6 to 6.5, and thetemperature was kept at 30° C. with positive dissolved oxygen.

[0212] Mycelial samples were harvested at 2, 4, 6, and 8 dayspost-inoculum and quick-frozen in liquid nitrogen. The samples werestored at −80° C. until they were disrupted for RNA extraction.

Example 2

[0213] cDNA Library Construction

[0214] Total cellular RNA was extracted from the mycelial samplesdescribed in Example 1 according to the method of Timberlake and Barnard(1981, Cell 26: 29-37), and the RNA samples were analyzed by Northernhybridization after blotting from 1% formaldehyde-agarose gels (Davis etal., 1986, Basic Methods in Molecular Biology, Elsevier SciencePublishing Co., Inc., New York). Polyadenylated mRNA fractions wereisolated from total RNA with an mRNA Separator Kit™ (ClontechLaboratories, Inc., Palo Alto, Calif.) according to the manufacturer'sinstructions. Double-stranded cDNA was synthesized using approximately 5μg of poly(A)+mRNA according to the method of Gubler and Hoffman (1983,Gene 25: 263-269) except a Notl-(dT)18 primer (Pharmacia Biotech, Inc.,Piscataway, N.J.) was used to initiate first strand synthesis. The cDNAwas treated with mung bean nuclease (Boehringer Mannheim Corporation,Indianapolis, Ind.) and the ends were made blunt with T4 DNA polymerase(New England Biolabs, Beverly, Mass.).

[0215] The cDNA was digested with Notl, size selected by agarose gelelectrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1 (InvitrogenCorporation, Carlsbad, Calif.) which had been cleaved with Notl plusEcoRV and dephosphorylated with calf-intestine alkaline phosphatase(Boehringer Mannheim Corporation, Indianapolis, Ind.). The ligationmixture was used to transform competent E. coli TOP10 cells (InvitrogenCorporation, Carlsbad, Calif.). Transformants were selected on 2YT agarplates (Miller, 1992, A Short Course in Bacterial Genetics. A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.) which contained kanamycinat a final concentration of 50 μg/ml.

[0216] Two independent directional cDNA libraries were constructed usingthe plasmid cloning vector pZErO-2.1. Library A was made using mRNA frommycelia harvested at four days, and Library B was constructed with mRNAfrom the six day time point. Neither cDNA library was amplified in orderto examine a representative “snapshot” of the gene expression profile inthe cells. Instead the libraries were plated, titered, and independentclones from each was analyzed by DNA sequencing.

[0217] Library A (4 day cells) consisted about 7.5×10⁴ independentclones and Library B (6 day cells) consisted of roughly 1.2×10⁵ clones.Miniprep DNA was isolated from forty colonies in each library andchecked for the presence and size of cDNA inserts. In this analysis 39of 40 colonies (97.5%) from Library A contained inserts with sizesranging from 600 bp to 2200 bp (avg.=1050 bp). Similarly, 39 of 40colonies (97.5%) picked from Library B had inserts with sizes rangingfrom 800 bp to 3600 bp (avg.=1380 bp).

Example 3

[0218] Template Preparation and Nucleotide Sequencing

[0219] From each cDNA library described in Example 2, 1192 transformantcolonies were picked directly from the transformation plates into96-well microtiter dishes which contained 200 μl of 2YT broth (Miller,1992, supra) with 50 μg/ml kanamycin. The plates were incubatedovernight at 37° C. without shaking. After incubation 100 μl of sterile50% glycerol was added to each well. The transformants were replicatedinto secondary, deep-dish 96-well microculture plates (Advanced GeneticTechnologies Corporation, Gaithersburg, Md.) containing 1 ml ofMagnificent Broth™ (MacConnell Research, San Diego, Calif.) supplementedwith 50 μg of kanamycin per ml in each well. The primary microtiterplates were stored frozen at −80° C. The secondary deep-dish plates wereincubated at 37° C. overnight with vigorous agitation (300 rpm) onrotary shaker. To prevent spilling and cross-contamination, and to allowsufficient aeration, each secondary culture plate was covered with apolypropylene pad (Advanced Genetic Technologies Corporation,Gaithersburg, Md.) and a plastic microtiter dish cover.

[0220] DNA was isolated from each well using the 96-well Miniprep Kitprotocol of Advanced Genetic Technologies Corporation (Gaithersburg,Md.) as modified by Utterback et al. (1995, Genome Sci. Technol. 1:1-8). Single-pass DNA sequencing was done with a Perkin-Elmer AppliedBiosystems Model 377 XL Automatic DNA Sequencer (Perkin-Elmer AppliedBiosystems, Inc., Foster City, Calif.) using dye-terminator chemistry(Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and thereverse lac sequencing primer.

Example 4

[0221] Analysis of DNA Sequence Data

[0222] Nucleotide sequence data were scrutinized for quality, andsamples giving improper spacing or ambiguity levels exceeding 2% werediscarded or re-run. Vector sequences were trimmed manually withassistance of FACTURA™ software (Perkin-Elmer Applied Biosystems, Inc.,Foster City, Calif.). In addition, sequences were truncated at the endof each sample when the number of ambiguous base calls increased. Allsequences were compared to each other to determine multiplicity usingAutoAssembler™ software (Perkin-Elmer Applied Biosystems, Inc., FosterCity, Calif.). Lastly, all sequences were translated in three frames andsearched against a non-redundant data base (NRDB) using GeneAssist™software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.)with a modified Smith-Waterman algorithm using the BLOSUM 62 matrix witha threshold score of 70. The NRDB was assembled from Genpept,Swiss-Prot, and PIR databases.

Example 5

[0223] Identification of lactonohydrolase cDNA Clone

[0224] Putative lactonohydrolase clones were identified by partialsequencing of random cDNA clones using an Applied Biosystems Model 377XL Automated DNA Sequencer according to the manufacturer's instructionsand comparison of the deduced amino acid sequence to the amino acidsequence of Fusarium oxysporum lactonohydrolase (Swissprot accessionnumber W21857) as described in Example 4. Among several clonesdiscovered in this manner, one was presumed to be full-length on thebasis of its alignment to the Fusarium oxysporum lactonohydrolase aminoacid sequence and the presence of a possible signal peptide, detectedusing the Signal-P computer program (Nielsen, et al., 1997, ProteinEngineering 10: 1-6). This clone designated E. coli FA0576, containingpFA0576 was selected for nucleotide sequence analysis and expressionstudies.

Example 6

[0225] Nucleotide sequencing and characterization of the Fusariumvenenatum lactonohydrolase cDNA from E. coli FA0576

[0226] DNA sequencing was performed with an Applied Biosystems Model 377XL Automated DNA Sequencer using dye-terminator chemistry. Contiguoussequences were generated using a transposon insertion strategy (PrimerIsland Transposition Kit, Perkin-Elmer/Applied Biosystems, Inc., FosterCity, Calif.). The lactonohydrolase clone from E. coli FA0576 wassequenced to an average redundancy of 5.9.

[0227] The lactonohydrolase clone encoded an open reading frame of 1200bp encoding a polypeptide of 400 amino acids. The nucleotide sequence(SEQ ID NO. 1) and deduced amino acid sequence (SEQ ID NO. 2) are shownin FIG. 1. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 17 residues was predicted.Thus, the mature lactonohydrolase is composed of 383 amino acids.

[0228] A comparative alignment of lactonohydrolase sequences wasundertaken using the Clustal method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10, and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5.

[0229] The comparative alignment showed that the Fusarium venenatumlactonohydrolase shares 87% identity with the lactonohydrolase fromFusarium oxysporum (WO 97/10341). There are 5 potential N-linkedglycosylation sites (Asn-X-Ser/Thr) within the Fusarium venenatumlactonohydrolase, however, one of these sites contains an internal Proresidue, and thus, it is not likely to be utilized. All of the remainingglycosylation sites are conserved in the Fusarium oxysporumlactonohydrolase, which is known to bear 3 types of N-linkedhigh-mannose carbohydrates at Asn residues 28, 106, 179, and 277 (WO97/10341).

Example 7

[0230] Construction of pDM181

[0231] Plasmid pDM181 was constructed using the technique of spliceoverlap extension to fuse the 1.2 kb Fusarium oxysporum trypsin promoter(SP387) to the 1.1 kb Fusarium oxysporum trypsin terminator (SP387). Apolylinker containing SwaI, KpnI and Pacd restriction sites was insertedbetween the promoter and terminator as part of the overlapping PCRstrategy. At the 5′ end of the promoter a XhoI site was added and thenative EcoRI site was preserved. At the 3′ end of the terminator EcoRI,HindIII and NsiI sites were incorporated by the PCR reaction.

[0232] A PCR fragment containing −1208 to −1 of the Fusarium oxysporumtrypsin promoter plus a 25 base pair polylinker was generated fromplasmid pJRoy20 (Royer et al., 1995, Biotechnology 13: 1479-1483) usingthe following primers: Primer 1 (sense):5′-GAGCTCGAGGAATTCTTACAAACCTTCAAC-3′ (SEQ ID NO. 3)      XhoI  EcoRIPrimer 2 (antisense):5′-TTAATTAAGGTACCTGAATTTAAATGGTGAAGAGATAGATATCCAAG-3′ (SEQ ID NO. 4)    PacI     KpnI      SwaI

[0233] The 100 μl PCR reaction contained 1×Pwo buffer (BoehringerMannheim, Indianapolis, Ind.), 200 μM each of dATP, dCTP, dGTP, anddTTP,10 ng of pJRoy20, and 5 units of Pwo DNA polymerase (BoehringerMannheim, Indianapolis, Ind.). PCR conditions used were 95° C. for 3minutes followed by 25 cycles each at 95° C. for 30 seconds, 50° C. for1 minute, and 72° C. for 1 minute. The final extension cycle was at 72°C. for 5 minutes.

[0234] Using the same PCR conditions, a second PCR fragment containingbp −5 to −1 of the Fusarium oxysporum trypsin promoter, a 25 base pairpolylinker, and 1060 base pairs of the 3′ untranslated region of theFusarium oxysporum trypsin gene (terminator region) was generated fromplasmid pJRoy20 using the following primers: Primer 3 (sense):5′-TCACCATTTAAATTCAGGTACCTTAATTAAATTCCTTGTTGGAAGCGTCGA-3′ (SEQ ID NO. 5)         SwaI      KpnI    PacI Primer 4 (antisense):5′-TGGTATGCATAAGCTTGAATTCAGGTAAACAAGATATAATTT-3′ (SEQ ID NO. 6)       NsiI HindIII EcoRI

[0235] The final 2.3 kb overlapping PCR fragment which contained −1208to −1 of the Fusarium oxysporum trypsin promoter, the 25 base pairpolylinker and 1060 base pairs of the Fusarium oxysporum trypsinterminator was obtained using 0.2 μl of the first PCR (promoter)reaction and 3 μl of the second (terminator) reaction as templated andprimers 1 and 4. The PCR conditions used were 95° C. for 3 minutesfollowed by 30 cycles each at 95° C. for 30 seconds, 62° C. for 1minute, and 72° C. for 3 minutes. The final extension cycle was at 72°C. for 5 minutes. Pwo DNA polymerase was also used for this reaction.

[0236] The resulting 2.3 kb fragment containing the trypsin promoter,the polylinker, and the trypsin terminator was digested with EcoRI andligated into the EcoRI digested vector pMT1612 containing the bar gene(WO 97/26330) to create pDM181 (FIG. 2).

Example 8

[0237] Construction of Plasmid pSheB1

[0238] The Fusarium venenatum expression vector pSheB1 (FIG. 3) wasgenerated by modification of pDM181. The modifications included (a)removal of two Ncol sites within the pDM181 sequence, and (b)restoration of the natural translation start of the Fusarium oxysporumtrypsin promoter (reconstruction of an Ncol site at the ATG startcodon).

[0239] Removal of two Ncol sites within the pDM181 sequence wasaccomplished using the QuikChange™ site-directed mutagenesis kit(Stratagene Cloning Systems, La Jolla, Calif.) according to themanufacturer's instruction with the following pairs of mutagenesisprimers:

[0240] 5′-dCAGTGAATTGGCCTCGATGGCCGCGGCCGCGAATT-3′ plus (SEQ ID NO. 7)

[0241] 5′-dAATTCGCGGCCGCGGCCATCGAGGCCAATTCACTG-3′ (SEQ ID NO. 8)

[0242] 5′-dCACGAAGGAAAGACGATGGCTTTCACGGTGTCTG-3′ plus (SEQ ID NO. 9)

[0243] 5′-dCAGACACCGTGAAAGCCATCGTCTTTCCTTCGTG-3′ (SEQ ID NO. 10)

[0244] Restoration of the natural translation start of the Fusariumoxysporum trypsin promoter was also accomplished using the StratageneQuikChange™ site directed mutagenesis kit in conjunction with thefollowing pair of mutagenesis primers:

[0245] 5′-dCTATCTCTTCACCATGGTACCTTAATTAAATACCTTGTTGGAAGCG-3′ plus (SEQID NO. 11)

[0246] 5′-dCGCTTCCAACAAGGTATTTAATTAAGGTACCATGGTGAAGAGATAG-3′ (SEQ ID NO.12)

[0247] All site-directed changes were confirmed by DNA sequence analysisof the appropriate vector regions.

Example 9

[0248] Construction of Expression Vector pTriggs1

[0249] The lactonohydrolase-expression vector pTriggs1 (FIG. 4) usingthe following protocol. The lactonohydrolase coding region was amplifiedfrom clone FA0576 using the following pair of primers:

[0250] 5′-dCGGCATGCCTTCCACCATTGCTG-3′ (forward) (SEQ ID NO. 13)

[0251] 5′-dTTAATTAACTAGTCATATAACTTGGGTCC-3′ (reverse) (SEQ ID NO. 14).

[0252] The forward primer introduces an SphI restriction site at thestart codon, and the reverse primer introduces a PacI site after thestop codon.

[0253] The amplification reaction (50 μl) contained the followingcomponents: 0.8 μg of clone FA0576 cDNA, 40 pmol of the forward primer,40 pmol of the reverse primer, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1×Pwo DNA polymerase buffer, and 2.5 units of Pwo DNA polymerase.The reactions were incubated in a Perkin-Elmer Model 480 Thermal Cyclerprogrammed for 30 cycles each at 95° C. for 3 minutes, 58° C. for 2minutes, and 72° C. for 2 minutes. The reaction products were isolatedon a 1.5% agarose gel (Eastman Kodak, Rochester, N.Y.) where a 1.2 kbproduct band was excised from the gel and purified using Qiaex II(Qiagen, Chatsworth, Calif.) according to the manufacturer'sinstructions.

[0254] The amplified lactonohydrolase segment was digested with SphI andthen treated with DNA polymerase I (Klenow fragment; BoehringerMannheim, Indianapolis, Ind.) in the presence of dNTPs. The 3→5′exonuclease activity of this enzyme removes the 4 nucleotides of theSphI cohesive end, generating a blunt-ended DNA fragment. TheKlenow-treated fragment was then digested with PacI and purified byagarose gel electrophoresis using standard methods (see Sambrook et al.,1989, supra).

[0255] The purified DNA segment was ligated to the vector pSheB1 whichhad been previously cleaved with Ncol, treated with DNA polymerase I(Klenow fragment) as above, then digested with PacI. Treating theNcol-digested vector with Klenow fragment results in “filling-in” of theNcol cohesive end, thereby making it blunt and compatible with theKlenow-reated SphI site of the lactonohydrolase DNA segment. Theresulting expression plasmid was designated pTriggs1 (FIG. 4).

Example 10

[0256] Expression of Lactonohydrolase cDNA in Fusarium venenatum

[0257] Spores of Fusarium venenatum CC1-3 (MLY-3) were generated byinoculating a flask containing 500 ml of RA sporulation medium with 10plugs from a 1×Vogels medium plate (2.5% Noble agar) supplemented with2.5% glucose and 2.5 mM sodium nitrate and incubating at 28° C., 150 rpmfor 2 to 3 days. Spores were harvested through Miracloth (Calbiochem,San Diego, Calif.) and centrifuged 20 minutes at 7000 rpm in a SorvallRC-5B centrifuge (E. I. DuPont De Nemours and Co., Wilmington, Del.).Pelleted spores were washed twice with sterile distilled water,resuspended in a small volume of water, and then counted using ahemocytometer.

[0258] Protoplasts were prepared by inoculating 100 ml of YEPG mediumwith 4×10⁷ spores of Fusarium venenatum CC1-3 and incubating for 16hours at 24° C. and 150 rpm. The culture was centrifuged for 7 minutesat 3500 rpm in a Sorvall RT 6000D (E. I. DuPont De Nemours and Co.,Wilmington, Del.). Pellets were washed twice with 30 ml of 1 M MgSO₄ andresuspended in 15 ml of 5 mg/ml of NOVOZYME 234™ (batch PPM 4356, NovoNordisk A/S, Bagsværd, Denmark) in 1 M MgSO₄. Cultures were incubated at24° C. and 150 rpm until protoplasts formed. A volume of 35 ml of 2 Msorbitol was added to the protoplast digest and the mixture wascentrifuged at 2500 rpm for 10 minutes. The pellet was resuspended,washed twice with STC, and centrifuged at 2000 rpm for 10 minutes topellet the protoplasts. Protoplasts were counted with a hemocytometerand resuspended in an 8:2:0.1 solution of STC:SPTC:DMSO to a finalconcentration of 1.25×10⁷ protoplasts/ml. The protoplasts were stored at−80° C., after controlled-rate freezing in a Nalgene Cryo 1° C. FreezingContainer (VWR Scientific, Inc., San Francisco, Calif.).

[0259] Frozen protoplasts of Fusarium venenatum CC1-3 were thawed onice. Five μg of pTriggs1 described in Example 9 and 5 μl of heparin (5mg per ml of STC) was added to a 50 ml sterile polypropylene tube. Onehundred μl of protoplasts was added, mixed gently, and incubated on icefor 30 minutes. One ml of SPTC was added and incubated 20 minutes atroom temperature. After the addition of 25 ml of 40° C. COVE topagarose, the mixture was poured onto an empty 150 mm diameter plate andincubated overnight at room temperature. Then an additional 25 ml of 40°C. COVE top agarose containing 10 mg of BASTA™ per ml was poured on topof the plate and incubated at room temperature for up to 14 days. Theactive ingredient in the herbicide BASTA™ is phosphinothricin. BASTA™was obtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) and wasextracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), andonce with chloroform:isoamyl alcohol (24:1) before use.

[0260] Three transformants were picked directly from the selectionplates (COVE underlay with COVE-BASTA™ overlay) into 125 ml shake flaskscontaining 25 ml of M400Da medium supplemented with 1 mM CaCl₂ and 100μg/ml ampicillin (to prevent bacterial contamination) and incubated at28° C., 200 rpm on a platform shaker for 6 days. The untransformedrecipient strain was also included as a negative control.

[0261] Flasks were sampled at 6 days. Cells were removed bycentrifugation, and 10 μl of each supernatant sample was heated to 95°C. for 5 minutes with an equal volume of SDS-PAGE sample buffer (NovexExperimental Technology, San Diego, Calif.). The denatured supernatantproteins were separated on a 10-20% gradient gel (Novex ExperimentalTechnology, San Diego, Calif.) and stained with Coomassie blue

[0262] SDS-PAGE analysis showed that the lactonohydrolase-producingtransformants secrete a prominent polypeptide with an apparent molecularweight of approximately 55 kDa. The apparent molecular weight of thisspecies (ca. 55,000) is substantially greater than the predictedmolecular weight of the mature lactonohydrolase (ca. 41,600), suggestingthat the enzyme may be extensively glycosylated. The observed molecularweight agrees closely to the subunit molecular weight of the Fusariumoxysporum lactonohydrolase (60,000) reported by Shimizu and Kataoka(1996, Annals of the New York Academy of Sciences 799: 650-658). Activelactonohydrolase is reportedly a dimer (Shimizu and Kataoka, 1996,supra).

Example 11

[0263] Purification of Recombinantly Produced Lactonohydrolase

[0264] Shake flask broth obtained as described in Example 9 was filteredthrough Miracloth then frozen. The thawed sample was filtered through a0.45 μm syringe filter and diluted and pH adjusted to 2.4 mS and pH 7.5.The sample was loaded onto a Q-Sepharose Big Beads column (90 ml) whichhad been pre-equilibrated using 20 mM Tris-HCl pH 7.5, containing 1 mMCaCl₂. The column was washed until baseline absorbance was reached. Alinear gradient from 0-0.3 M NaCl in 20 mM Tris-HCl pH 7.5, containing 1mM CaCl₂ was run over 6.67 column volumes. Fractions were assayed usingthe following assay.

[0265] A 20 μl volume of enzyme sample was added to 140 μl of 50 mMpotassium phosphate pH 7.0 along with 40 μl of 100 mM D-galacturonicacid γ lactone in 50 mM potassium phosphate pH 7.0. The reaction wasincubated for 34 minutes. A 50 μl sample of the solution was placed intoanother well along with 50 μl of a 1:1 2 N hydroxylamine-HCl to 3.5 NNaOH solution. Then 100 μl of a 1:1 ethanol to 10% w/v ferric chloridein 4 N HCl was added. The absorbance at 520 nm was then measured. Adecrease in absorbance at 520 nm indicated a lower concentration of thelactone substrate.

[0266] The eluted enzyme was determined to be homogeneous by SDS PAGE.

Example 12

[0267] Lactonization of Pantoic Acid

[0268] Pantoic acid was prepared by hydrolyzing pantoyl lactone (130 mg)in 2 ml of 5 N NaOH for 15 minutes at room temperature. The solution pHwas adjusted to 5 by the addition of ˜1.5 ml 6M HCl and water was addedto a final volume of 10 ml to achieve a final pantoic acid concentrationof 100 mM.

[0269] The substrate was incubated with broth for 30 minutes at 30° C.and assayed for lactone formation by the standard method below.Incubation with 0.25 M sodium acetate pH 5 resulted in four-fold moreextensive lactonization than 20 mM sodium phosphate pH 6.4.

[0270] Deposit of Biological Material

[0271] The following biological material has been deposited under theterms of the Budapest Treaty with the Agricultural Research ServicePatent Culture Collection, Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, and given the followingaccession number: Deposit Accession Number Date of Deposit E. coli TOP10(pFA0576) NRRL B-30074 Oct. 27, 1998

[0272] The strain has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0273] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

[0274] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 14 1 1334 DNA Fusarium 1 ctcccacacc actcagtttc acttctacct cattcgccatgccttccacc attgctgccc 60 ttgttgtcgg gatttgtggc gttgccgctg ccaaacttccctccacggct caggtcattg 120 atcagaagtc tttcaatgtt ttgaaggatg tcccaccaccttccgtggct aatgacacac 180 tggtctttac atggcccgga gtgacagagg aatctctcgtcgagaaaccc ttccatgttt 240 atgatgatga attcctcgat gtcatcggaa aggatccctctctgacactt gttgctacgt 300 cagaaagcga ccccatcttc cacgaggctg tagtctggtacccacctaca gacgaggttt 360 ttttcgtgca aaatgcgggt gctcctgcgg caggcactggcctgaacaag tcttccatca 420 tccagaagat ttctctcaaa gatgcagagg ctttgcgcaagggaacccta ggcaaggatg 480 aagtgaaggt gacagtcgtt gacacagcta accctcaagtcattaacccc aatggtggca 540 tttactacaa gggcgaaatc atctttgctg gtgaaggccaaggtgacgaa gttccctcgg 600 ccctttaccg catgaacccc ttgcctccat acaacacaagcaccctcctc aacaactact 660 ttggccgcca gttcaactcc ttgaacgacg ttggcatcaaccccaggaat ggtgacttgt 720 acttcaccga cactctctac ggctatctcc aagacttccgtcctgtccct ggtctgcgaa 780 accaagtgta ccgatacaac ttcgacactg gtgctgtaactgttgtcgct gatgacttta 840 ctcttcccaa cggtattggt tttgctcctg atggaaagcgtgtctatgtc accgacactg 900 gcatcgctct tggcttctac ggccgtaacc tttcctcccccgcctctgtt tactctttcg 960 acgtgaacaa ggatggtacc cttgagaacc gcaagacttttgcctacgta gcttctttca 1020 tcccagacgg tgttcatacc gattccaagg gtcgtgtctatgctggttgt ggtgacggtg 1080 tccatgtctg gaacccctct ggcaagctca ttggcaagatctataccggg atcactgctg 1140 ccaacttcca atttgctgga aaaggaagat tgattatcactggtcagact aagctgttct 1200 acgttaccct ggctgcttca ggacccaagt tatatgactagaactcccct gtggcagtat 1260 agaaacagat attttaccgt attgatagaa gataatattaattattaatc gataaaaaaa 1320 aaaaaaaaaa aaaa 1334 2 400 PRT Fusarium 2 MetPro Ser Thr Ile Ala Ala Leu Val Val Gly Ile Cys Gly Val Ala 1 5 10 15Ala Ala Lys Leu Pro Ser Thr Ala Gln Val Ile Asp Gln Lys Ser Phe 20 25 30Asn Val Leu Lys Asp Val Pro Pro Pro Ser Val Ala Asn Asp Thr Leu 35 40 45Val Phe Thr Trp Pro Gly Val Thr Glu Glu Ser Leu Val Glu Lys Pro 50 55 60Phe His Val Tyr Asp Asp Glu Phe Leu Asp Val Ile Gly Lys Asp Pro 65 70 7580 Ser Leu Thr Leu Val Ala Thr Ser Glu Ser Asp Pro Ile Phe His Glu 85 9095 Ala Val Val Trp Tyr Pro Pro Thr Asp Glu Val Phe Phe Val Gln Asn 100105 110 Ala Gly Ala Pro Ala Ala Gly Thr Gly Leu Asn Lys Ser Ser Ile Ile115 120 125 Gln Lys Ile Ser Leu Lys Asp Ala Glu Ala Leu Arg Lys Gly ThrLeu 130 135 140 Gly Lys Asp Glu Val Lys Val Thr Val Val Asp Thr Ala AsnPro Gln 145 150 155 160 Val Ile Asn Pro Asn Gly Gly Ile Tyr Tyr Lys GlyGlu Ile Ile Phe 165 170 175 Ala Gly Glu Gly Gln Gly Asp Glu Val Pro SerAla Leu Tyr Arg Met 180 185 190 Asn Pro Leu Pro Pro Tyr Asn Thr Ser ThrLeu Leu Asn Asn Tyr Phe 195 200 205 Gly Arg Gln Phe Asn Ser Leu Asn AspVal Gly Ile Asn Pro Arg Asn 210 215 220 Gly Asp Leu Tyr Phe Thr Asp ThrLeu Tyr Gly Tyr Leu Gln Asp Phe 225 230 235 240 Arg Pro Val Pro Gly LeuArg Asn Gln Val Tyr Arg Tyr Asn Phe Asp 245 250 255 Thr Gly Ala Val ThrVal Val Ala Asp Asp Phe Thr Leu Pro Asn Gly 260 265 270 Ile Gly Phe AlaPro Asp Gly Lys Arg Val Tyr Val Thr Asp Thr Gly 275 280 285 Ile Ala LeuGly Phe Tyr Gly Arg Asn Leu Ser Ser Pro Ala Ser Val 290 295 300 Tyr SerPhe Asp Val Asn Lys Asp Gly Thr Leu Glu Asn Arg Lys Thr 305 310 315 320Phe Ala Tyr Val Ala Ser Phe Ile Pro Asp Gly Val His Thr Asp Ser 325 330335 Lys Gly Arg Val Tyr Ala Gly Cys Gly Asp Gly Val His Val Trp Asn 340345 350 Pro Ser Gly Lys Leu Ile Gly Lys Ile Tyr Thr Gly Ile Thr Ala Ala355 360 365 Asn Phe Gln Phe Ala Gly Lys Gly Arg Leu Ile Ile Thr Gly GlnThr 370 375 380 Lys Leu Phe Tyr Val Thr Leu Ala Ala Ser Gly Pro Lys LeuTyr Asp 385 390 395 400 3 30 DNA Fusarium 3 gagctcgagg aattcttacaaaccttcaac 30 4 47 DNA Fusarium 4 ttaattaagg tacctgaatt taaatggtgaagagatagat atccaag 47 5 51 DNA Fusarium 5 tcaccattta aattcaggtaccttaattaa attccttgtt ggaagcgtcg a 51 6 42 DNA Fusarium 6 tggtatgcataagcttgaat tcaggtaaac aagatataat tt 42 7 35 DNA Fusarium 7 cagtgaattggcctcgatgg ccgcggccgc gaatt 35 8 35 DNA Fusarium 8 aattcgcggc cgcggccatcgaggccaatt cactg 35 9 34 DNA Fusarium 9 cacgaaggaa agacgatggc tttcacggtgtctg 34 10 34 DNA Fusarium 10 cagacaccgt gaaagccatc gtctttcctt cgtg 3411 46 DNA Fusarium 11 ctatctcttc accatggtac cttaattaaa taccttgttg gaagcg46 12 46 DNA Fusarium 12 cgcttccaac aaggtattta attaaggtac catggtgaagagatag 46 13 23 DNA Fusarium 13 cggcatgcct tccaccattg ctg 23 14 29 DNAFusarium 14 ttaattaact agtcatataa cttgggtcc 29

What is claimed is:
 1. An isolated polypeptide having lactonohydrolaseactivity, selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 95% identity with aminoacids 18 to 400 of SEQ ID NO. 2; (b) a polypeptide encoded by a nucleicacid sequence having at least 95% homology with nucleotides 90 to 1238of SEQ ID NO. 1; (c) a variant of the polypeptide having an amino acidsequence of SEQ ID NO. 2 comprising a substitution, deletion, and/orinsertion of one or more amino acids; (d) an allelic variant of (a) or(b); and (e) a fragment of (a), (b), or (d) that has lactonohydrolaseactivity.
 2. The polypeptide of claim 1, having an amino acid sequencewhich has at least 95% identity with amino acids 18 to 400 of SEQ ID NO.2.
 3. The polypeptide of claim 2, having an amino acid sequence whichhas at least 97% identity with amino acids 18 to 400 of SEQ ID NO.
 2. 4.The polypeptide of any of claims 1-3, comprising the amino acid sequenceof SEQ ID NO.
 2. 5. The polypeptide of any of claims 1-4, consisting ofthe amino acid sequence of SEQ ID NO. 2 or a fragment thereof.
 6. Thepolypeptide of claim 5, consisting of the amino acid sequence of SEQ IDNO.
 2. 7. The polypeptide of claim 6, which consists of amino acids 18to 400 of SEQ ID NO.
 2. 8. The polypeptide of claim 1, which is encodedby a nucleic acid sequence having at least 95% homology with nucleotides90 to 1238 of SEQ ID NO.
 1. 9. The polypeptide of claim 8, which isencoded by a nucleic acid sequence having at least 97% homology withnucleotides 90 to 1238 of SEQ ID NO.
 1. 10. The polypeptide of claim 1,wherein the polypeptide is a variant of the polypeptide having an aminoacid sequence of SEQ ID NO. 2 comprising a substitution, deletion,and/or insertion of one or more amino acids.
 11. The polypeptide ofclaim 1, which is encoded by the nucleic acid sequence contained inplasmid pFA0576 which is contained in E. coli NRRL B-30074.
 12. Thepolypeptide of any of claims 1-11 which has at least 20% of thelactonohydrolase activity of SEQ ID NO.
 2. 13. A polypeptide having thesame lactonohydrolase activity as the polypeptide of any of claims 1-12.14. An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes the polypeptide of any of claims 1-13.
 15. An isolatednucleic acid sequence comprising a nucleic acid sequence having at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO. 1,in which the mutant nucleic acid sequence encodes a polypeptideconsisting of amino acids 18 to 400 of SEQ ID NO.
 2. 16. A nucleic acidconstruct comprising the nucleic acid sequence of claim 14 operablylinked to one or more control sequences that direct the production ofthe polypeptide in a suitable expression host.
 17. A recombinantexpression vector comprising the nucleic acid construct of claim
 16. 18.A recombinant host cell comprising the nucleic acid construct of claim16.
 19. A method for producing a mutant nucleic acid sequence,comprising (a) introducing at least one mutation into the maturepolypeptide coding sequence of SEQ ID NO. 1, wherein the mutant nucleicacid sequence encodes a polypeptide consisting of amino acids 18 to 400of SEQ ID NO. 2; and (b) recovering the mutant nucleic acid sequence.20. A mutant nucleic acid sequence produced by the method of claim 19.21. A method for producing a polypeptide, comprising (a) cultivating astrain comprising the mutant nucleic acid sequence of claim 20 encodingthe polypeptide to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide.
 22. A method for producing thepolypeptide of any of claims 1-13 comprising (a) cultivating a strain toproduce a supernatant comprising the polypeptide; and (b) recovering thepolypeptide.
 23. A method for producing the polypeptide of any of claims1-13 comprising (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleic acid sequence encoding the polypeptideunder conditions suitable for production of the polypeptide; and (b)recovering the polypeptide.
 24. A method for producing a polypeptidecomprising (a) cultivating a host cell under conditions conducive forproduction of the polypeptide, wherein the host cell comprises a mutantnucleic acid sequence having at least one mutation in the maturepolypeptide coding sequence of SEQ ID NO. 1, wherein the mutant nucleicacid sequence encodes a polypeptide consisting of amino acids 18 to 400of SEQ ID NO. 2, and (b) recovering the polypeptide.
 25. A method forproducing the polypeptide of any of claims 1-13 comprising (a)cultivating a homologously recombinant cell, having incorporated thereina new transcription unit comprising a regulatory sequence, an exon,and/or a splice donor site operably linked to a second exon of anendogenous nucleic acid sequence encoding the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 26. A method for producing a mutant of acell, which comprises disrupting or deleting a nucleic acid sequenceencoding the polypeptide of any of claims 1-13 or a control sequencethereof, which results in the mutant producing less of the polypeptidethan the cell.
 27. A mutant produced by the method of claim
 26. 28. Themutant of claim 27, which further comprises a nucleic acid sequenceencoding a heterologous protein.
 29. A method for producing aheterologous polypeptide comprising (a) cultivating the mutant of claim28 under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 30. A nucleic acid construct comprising agene encoding a protein operably linked to a nucleic acid sequenceencoding a signal peptide consisting of nucleotides 38 to 89 of SEQ IDNO. 1, wherein the gene is foreign to the nucleic acid sequence.
 31. Arecombinant expression vector comprising the nucleic acid construct ofclaim
 30. 32. A recombinant host cell comprising the nucleic acidconstruct of claim
 30. 33. A method for producing a protein comprising(a) cultivating the recombinant host cell of claim 32 under conditionssuitable for production of the protein; and (b) recovering the protein.34. A method for preventing biofilm development on a liquid-solidinterface by one or more microorganisms, comprising administering aneffective amount of a composition comprising one or more polypeptideshaving lactonohydrolase activity and a carrier to the liquid-solidinterface to degrade one or more lactones produced by the one or moremicroorganisms, wherein the one or more lactones are involved in theformation of the biofilm.